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(Received for publication, September 13,
1995; and in revised form, January 17, 1996) From the
Nucleic acid specificity was tested for two monoclonal
anti-double-stranded DNA autoantibodies, 2C10 and H241, derived from
two lupus-prone MRL/Mp-lpr/lpr mice. Antibody 2C10 bound
double-stranded oligonucleotides with a preference for dA-dT over dG-dC
base pairs and did not bind single-stranded oligonucleotides.
Distamycin A, an antibiotic that binds to the minor groove, inhibited
2C10 binding of double-stranded DNA, suggesting that this antibody
interacts with dA-dT base pairs in the minor groove. Antibody H241
binding was previously shown to have a dG-dC preference and to involve
both major and minor grooves. In attempted footprinting assays, both
2C10 and H241 markedly enhanced rather than protected against cleavage
of DNA by hydroxyl radical-generating systems. With 2C10, this
enhancement effect was observed only when hydroxyl radical generation
was associated with oxidation of Fe(II). In contrast, H241 enhancement
occurred in the presence of H
Autoantibodies to dsDNA ( Because some, but not all, anti-DNA
antibodies are pathogenic, there has been much interest in whether the
pathogenicity is related to binding specificity, either for DNA
epitopes or for cross-reactive structures. Thus, many anti-DNA
autoantibodies have been analyzed extensively at the level of primary
structure and modeling of their binding sites (13, 14) or, when possible, by x-ray
crystallography(15, 16) . In addition, DNA-binding
sites of monoclonal anti-DNA antibodies and epitopes have been mapped
by competitive immunoassay with polynucleotides or
oligonucleotides(17, 18) . High resolution
footprinting, measuring protection against chemical cleavage of DNA,
has been useful in identifying the epitopes for autoantibodies to
single-stranded DNA (19) and experimentally induced antibodies
to Z-DNA (20) . In a previous study, we had examined the
specificity of a pathogenic monoclonal IgG anti-dsDNA antibody, H241,
derived from an MRL/Mp-lpr/lpr lupus mouse. In competitive
immunoassays with a series of synthetic double-stranded
oligonucleotides, H241 bound to a (dG-dC)
Figure 4:
Enhancement of hydroxyl radical cleavage
of DNA by anti-dsDNA antibodies. The 274-bp dsDNA probe was incubated
with anti-dsDNA antibody 2C10 (A) or H241 (B) or with
a control myeloma protein, MPC11, for 30 min in PBS. Then a 100
µM concentration of the divalent metal ion indicated at
the top of each lane was added. Four min later, an excess of
Na
Figure 5:
Dependence of the enhanced DNA cleavage on
the hydroxyl radical concentration. The DNA probe was cleaved by
Udenfriend's system (28) with anti-dsDNA antibody 2C10 (A) or H241 (B) or with a control myeloma protein,
MPC11. The standard reaction mixture (1
Figure 6:
Time course of the enhanced DNA cleavage
in the presence of anti-dsDNA antibodies. A, the DNA probe was
incubated with anti-dsDNA antibody 2C10 or H241 or with a control
myeloma protein, MPC11, and then cleaved by the reaction with the
standard concentration of Udenfriend's system reagents (28) for continuous hydroxyl radical generation along with
oxidation of Fe(EDTA)
Figure 7:
Inhibition of the DNA cleavage enhancement
effect of anti-dsDNA antibodies by oligonucleotides. Oligonucleotide K5
(5`-AAAATATATATTT-3`) or K6 (5`-GGGGCGCGCGCCC-3`) was added to the DNA
cleavage reaction using Udenfriend's system (28) in the
presence of anti-dsDNA antibodies 2C10 and H241 or a control myeloma
protein, MPC11. Based on the densitometric estimation of the amounts of
the starting materials, percent inhibition was calculated as follows:
inhibition = (1 - ((cleavage with anti-DNA and inhibitor)
- (cleavage with MPC11))/((cleavage with anti-DNA) -
(cleavage with MPC11)))
Figure 8:
DNA cleavage pattern enhanced by anti-DNA
antibodies. A, the DNA probe was cleaved by hydroxyl radical
generated by reduction of H
Figure 9:
Pattern of DNA cleavage by hydroxyl
radical produced along with oxidation of Cu(I) in the presence of 2C10.
The ordinate represents the relative sensitivity to the
cleavage calculated as described for Fig. 8. DNA was incubated
for 4 min in a mixture of 1 µM copper sulfate, 0.1 mM sodium ascorbate, and 0.9 mM H
Figure 1:
Competitive ELISA for 2C10. A,
antibody 2C10 was preincubated with various double- or single-stranded
oligonucleotides, and the mixture was added to microtiter plate wells
coated with native calf DNA.
For further experiments, we
used a 274-bp dsDNA probe purified from a modified plasmid as described
under ``Materials and Methods.'' First we confirmed the
binding of the antibodies to this DNA probe with an assay for
retardation of migration in gel electrophoresis. Antibody 2C10 shifted
the mobility of the
Figure 2:
Gel retardation assay for the binding of
anti-dsDNA antibodies to the 274-bp dsDNA probe. DNA was incubated with
anti-dsDNA antibody 2C10 (A) or H241 (B) or with a
control myeloma protein, MPC11, and loaded on a 10% polyacrylamide gel
with a 4% stacking gel. Molar ratios of DNA to antibody are indicated
at the top of each lane.
Figure 3:
Effect of distamycin A on the binding of
2C10 and H241 to dsDNA. Antibodies 2C10 (lanes 2-5) and
H241 (lanes 7-10) were incubated with the 274-bp dsDNA
probe alone at DNA/antibody ratios of 1:40 (lanes 2 and 7) and 1:80 (lanes 4 and 9) or with a
DNA/distamycin A mixture at DNA/antibody ratios of 1:40 (lanes 3 and 8) and 1:80 (lanes 5 and 10). The
mixtures were then loaded on a 10% polyacrylamide gel with a 4%
stacking gel. Lanes 1 and 6 are the DNA without
antibodies.
Preincubation of the 274-bp DNA probe
with distamycin A, an antibiotic that binds in the minor groove with
preference for dA-dT-rich sequences (27) , caused marked
inhibition of antibody 2C10 binding, but did not affect H241 binding (Fig. 3). Together with ELISA data described above, this result
suggests that 2C10 recognizes double helical structure of DNA and makes
contacts preferentially with regions of dA-dT base pairs in or over the
minor groove.
A mixture of Fe(II)
chelated with Na
The
next experiment measured the time dependence of this effect of anti-DNA
antibodies in the standard reaction mixture (Fig. 6). The
reaction was rapid in the presence of H241; >50% of the starting DNA
was degraded within 15 s. The reaction with 2C10 was slower under this
assay condition, but there was still significant enhancement of
cleavage over a 2-8-min period.
As a further test of this
interpretation, the extents of cleavage at specific sites in the DNA
sequence were compared in the analytical sequencing gels (Fig. 8). The susceptibility for hydroxyl radical cleavage in
the presence of 2C10 and H241 showed a reciprocal pattern. The
enhancement with antibody 2C10, relative to the reaction in the absence
of antibody, was greatest in dA-dT-rich sequences, whereas the
enhancement with antibody H241 was greatest in dG-dC-rich sequences,
again indicating that increased susceptibility for cleavage is related
to the specific binding sites of each antibody. In this experiment,
>95% of the DNA was uncut following the hydroxyl radical reaction
with a control antibody, MPC11. This condition is consistent with
single-hit kinetics(29) , supporting the conclusion that the
result truly reflects substrate specificity.
These experiments indicate that two anti-DNA autoantibodies
from lupus mice enhance the degradation of DNA by hydroxyl
radical-generating systems. Steps were taken to test whether the effect
was truly due to the antibody rather than a contaminant that might have
been present in an immune complex. The antibodies were highly purified,
as described under ``Materials and Methods.'' Their purity
was confirmed by SDS-polyacrylamide gel electrophoresis, which detected
no contamination by other DNA-binding proteins. Further verification
was obtained by counting the number of IgG-nucleosome complexes or free
nucleosomal particles among IgG particles in a fixed area in an
electron micrograph of a preparation that included mica flakes for
quick-freezing and deep-etching. In the case of monoclonal anti-DNA
antibodies obtained after a wash with a high salt concentration,
neither IgG-nucleosome complexes nor free nucleosomes were found among
80 or more IgG particles, whereas 42 out of 80 IgG particles were
associated with nucleosomes in the case of monoclonal antibodies
obtained by the conventional affinity chromatography without a high
salt wash. ( Specificity of the effects provided more direct evidence for the
role of the antibody-binding sites in this phenomenon. The relative
effectiveness of oligonucleotides as inhibitors of the enhancement
corresponded with their effectiveness as inhibitors of DNA binding.
Both the cleavage enhancement and DNA binding by antibody 2C10 were
inhibited preferentially by the AT oligonucleotide, and both activities
of H241 were inhibited preferentially by the GC oligonucleotide.
Furthermore, enhancement occurred preferentially at dA-dT segments of
the test DNA with antibody 2C10, but at dG-dC segments with H241. The
sequence specificity was not absolute, and neither were the binding
differences(21) . The effect was not due to uncontrolled
conditions of the hydroxyl-generating system; unlike the antibodies,
the Cro protein protected its specific target under the same
conditions, just as expected in a footprinting assay. Extensive
studies have demonstrated that hydroxyl radical generated by the Fenton
reaction modifies bases and cleaves
DNA(33, 34, 35, 36) . The ability of
bound protein to protect against this cleavage is the basis of high
resolution footprinting assays in
vitro(19, 26) , and protection against oxidative
species is afforded within cells by histones in condensed
chromatin(37) . On the other hand, DNA-nuclear matrix protein
association presents hypersensitive sites for oxidative damage,
possibly related to the binding of copper
ions(37, 38) . Metal-binding proteins such as ferritin (39) also mediate increased DNA cleavage, as can lactoferrin
with copper bound to its surface(40) , an inorganic polymer
that binds both iron and DNA (41) or small DNA-binding
molecules such as adriamycin(42) . These proteins, polymers,
and small molecules may focus the Fenton reaction on simultaneously
bound DNA. In addition, binding of iron to DNA may determine cleavage
sites(34) . It is not known how antibodies 2C10 and H241
enhanced DNA cleavage, but there were differences in the mechanisms
operating with the two antibodies. Because the enhancement with
antibody 2C10 occurred either with simple addition of Fe(II) to the
DNA-antibody complexes or with Fe(II) and the
H Antibody H241 acted
differently, as it was effective with H Because an excess of antibody was present in the solution and the
precise number of cleavage events was not known, it is not clear
whether the rate enhancement involved turnover, as in enzyme catalysis,
or whether it was stoichiometric. The antibodies alone did not
hydrolyze DNA in the time course of these reactions, up to 30 min.
Hydrolysis of DNA by purified IgG and Fab fragments from systemic lupus
erythematosus patients' sera has been detected by sensitive
assays (11, 12) . In addition, Paul and co-workers
have reported hydrolysis of vasointestinal peptide by IgG
autoantibodies and their Fab fragments (44, 45) or
isolated light chains (46) and by the purified (47) or
recombinant (48) light chain of a monoclonal antibody from a
mouse immunized with this peptide. Additional study will determine the
extent to which autoantibodies provide new approaches to isolation of
catalytic antibodies, adding to the large number that have been induced
with haptens based on transition state analogues (49) or
obtained by selection from combinatorial libraries(50) . Antibody that increases the sensitivity of DNA to cleavage by
hydroxyl radical or other reactive oxygen species could have an effect in vivo in sites of inflammation, where such species are
generated. For example, higher than normal oxygen radical production
has been detected in both bronchial alveolar cells and
polymorphonuclear cells from patients with systemic lupus
erythematosus(51) . Increased numbers of chromosomal breaks and
increased sensitivity to UV light, with involvement of reactive
species, have also been detected and suggested as a basis for the well
known sensitivity of these patients to UV
irradiation(52, 53) . Protection by superoxide
dismutase provided evidence that reactive oxygen species were involved
in the damage by UV radiation(52, 54) . The presence
of anti-DNA antibodies such as 2C10 or H241 could enhance the
sensitivity of DNA to such damage. In turn, hydroxyl radical-mediated
damage to DNA may increase the binding by anti-DNA antibodies (55, 56) and increase the immunogenicity of DNA (56) .
Volume 271,
Number 11,
Issue of March 15, 1996 pp. 6555-6561
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
O and ascorbate or
UV light irradiation and did not depend on added metal ion. The
enhancement sites were related to the antibody binding specificities.
The oligonucleotide 5`-AAAATATATATTT-3` was a much more effective
inhibitor of the 2C10 enhancement than of the H241 effect, whereas the
oligonucleotide 5`-GGGGCGCGCGCCC-3` was a much more effective inhibitor
of the H241 enhancement. In addition, the enhanced cleavage occurred
preferentially at dA-dT-rich regions with 2C10 and at dG-dC-rich
regions with H241. These findings raise the possibility that anti-DNA
autoantibodies could enhance DNA damage in inflammatory lesions in
which hydroxyl radicals are generated.
)are characteristic of the
autoimmune disease systemic lupus erythematosus. Increased production
of anti-dsDNA antibodies reflects or predicts periods of active
clinical disease(1) , and anti-DNA antibodies can contribute to
pathogenesis of lesions. There are several possible bases for their
contribution to tissue damage. Anti-DNA antibodies are concentrated in
immune complexes in glomerular lesions of lupus nephritis, where they
may initiate inflammatory reactions(2, 3) . It was
originally proposed that glomerular lesions resulted from deposition of
DNA
anti-DNA complexes that formed in the circulation (2, 3) , but many anti-DNA antibodies cross-react with
other negatively charged polymers or membrane proteins (4, 5, 6) and may bind directly to structures
present in the glomerular basement membrane(4, 7) . As
well as initiating inflammation, anti-DNA antibodies may cause cell
damage more directly. Some anti-DNA antibodies bind to cell surfaces
and initiate complement-dependent cytotoxicity(8) , whereas
others penetrate the membrane of living cells and may reach the nucleus (8, 9, 10) . It has also been reported that
some anti-DNA autoantibodies present in sera of systemic lupus
erythematosus patients can catalyze the hydrolytic cleavage of
DNA(11, 12) .
or (dG-dC)
core in the center of a base-paired
octadecanucleotide(21) . This selectivity was also reflected in
the very marked preference of this antibody for poly(dG-dC) over
poly(dA-dT). A second IgG antibody, 2C10, isolated from a different
MRL/Mp-lpr/lpr mouse, had a very different selectivity,
markedly favoring poly(dA-dT) over poly(dG-dC)(22) .
Competitive assays of antibody 2C10 with synthetic oligonucleotides
indicated that it could bind well to alternating dA-dT, to stretches of
(dA)(dT), or to
(dA-dG)
(dC-dT) sequences, ()so it was more difficult
to assign a single kind of epitope to it. Recently, we tried to apply a
DNA footprinting assay to observe the DNA sites recognized by these two
monoclonal anti-dsDNA antibodies more directly. Unexpectedly, we found
that these two anti-dsDNA antibodies enhanced rather than protected
against the cleavage of DNA by hydroxyl radical-generating systems. The
two antibodies differed in the conditions under which they were
effective and in the sites of DNA cleavage they supported.
Purification of Monoclonal Anti-DNA
Antibodies
Monoclonal anti-dsDNA antibodies 2C10 and H241 (both
IgG2b,
) are hybridoma products derived from two lupus-prone
MRL/Mp-lpr/lpr mice (21, 22) . Hybridoma
cells were cultured in serum-free medium (Hymedium 606, Kohjin Bio,
Sakato, Japan), and the culture supernatants were loaded on a matrix
bearing immobilized staphylococcal protein A (Chromatotop Super Protein
A, Nihon Gaishi, Handa, Japan). The matrix was washed with 25 mM Tris-HCl, 140 mM NaCl, pH 7.4, followed by 1.5 M NaCl, 25 mM Tris-HCl, pH 7.4, in order to remove DNA and
DNA-binding proteins that may have attached to antibodies during
culture and isolation; the antibodies remained bound to the protein A.
Then the antibodies were recovered with elution buffer (250 mM glycine HCl, pH 3.5) and dialyzed immediately against PBS (8.1
mM NaHPO, 1.5 mM KHPO, 2.7 mM KCl, 137 mM NaCl, pH 7.3). Purity of the eluates was confirmed by
SDS-polyacrylamide gel electrophoresis, agarose gel electrophoresis,
and electron microscopy, which demonstrated that washing the column
with a solution of high ionic strength did remove bound antigens; no
contamination of DNA or DNA-binding proteins was detectable. ()A mouse myeloma protein, MPC11 (IgG2b,), which does
not bind to DNA, was similarly purified with the protein A matrix and
served as an isotype-matched control IgG.ELISA
UV-irradiated polystyrene microtiter plates
(Immulon 1, Dynatech Laboratories Inc., Chantilly, VA) (23) were coated with calf thymus native DNA, and competitive
assays using a series of synthetic oligonucleotides were performed as
described previously (21) .Preparation of a DNA Probe for Hydroxyl Radical
Footprinting
A self-complementary oligonucleotide that contains
alternating dA-dT and dG-dC segments
(5`-CGCGCGATATATATATCGCGCGATATATATATCGCGCG-3`) was inserted into the BamHI site of a pUC19 plasmid vector (Takara, Kyoto, Japan).
After amplification in Escherichia coli DH5
(Life
Technologies, Inc.), the plasmid was recovered by an alkaline lysis
miniprep procedure using a QIAGEN plamid kit and cleaved with EcoRI (Takara). The 5`-end of the EcoRI digestion
product was dephosphorylated with calf intestine alkaline phosphatase
(Boehringer Mannheim, Tokyo) following a standard
protocol(24) , and the oligonucleotide was purified with a spin
column (QIAquick spin polymerase chain reaction purification kit,
QIAGEN Inc., Chatsworth, CA) following the manufacturer's
protocol. This product was treated with PvuII (Takara), and a
274-bp fragment containing a 4-base protruding end was purified by 2%
agarose gel electrophoresis. This procedure allowed subsequent P labeling by T4 polynucleotide kinase (Stratagene, La
Jolla, CA) (24) at the end of just one strand.
Gel Retardation Assay
Mixtures of 7.2 ng of the
274-bp DNA probe and various quantities of antibodies in PBS were
incubated for 30 min at room temperature and analyzed by
electrophoresis in a 10% polyacrylamide gel with a 4% stacking gel as
described previously (25) . In an experiment to test
involvement of the DNA minor groove in binding of anti-DNA antibodies,
7.2 ng of the DNA probe was preincubated with 310 ng of distamycin A
(Sigma) for 20 min at room temperature before the reaction with
anti-DNA antibodies.Hydroxyl Radical Cleavage of DNA
All buffers were
prepared with deionized, distilled, and autoclaved water. The
5`-end-labeled 274-bp DNA probe, described above, was mixed with one of
the monoclonal anti-dsDNA antibodies or a control IgG in 8 µl of
PBS and incubated for 30 min at room temperature. In the experiments
shown in Fig. 4(A and B), 1 µl of freshly
prepared aqueous solution of ferrous ammonium sulfate, zinc sulfate,
magnesium chloride, calcium chloride, or copper sulfate (100 µM final concentration) was added to the DNA/antibody mixture and
allowed to react for 4 min at 25 °C. In the experiments shown in Fig. 4(C and D) and in Fig. 5Fig. 6Fig. 7Fig. 8, one of the metal
ions indicated in the legends was mixed with NaEDTA; then
the metal ion-EDTA complex was mixed with sodium ascorbate and
HO and added immediately to the DNA/antibody
mixture according to the protocol of Tullius et al.(26) with modifications. Final concentrations of metal ion,
EDTA, ascorbate, and HO in the standard
reaction mixture were 10 µM, 20 µM, 1
mM, and 9 mM, respectively. Concentrations were
changed as indicated for some experiments. The reaction time was 4 min
at 25 °C unless otherwise indicated. In all cases, the reactions
were quenched by addition of thiourea (to 0.1 M) and
NaEDTA (to 0.2 M). After addition of 0.3 M sodium acetate and 2.75 µg/µl yeast tRNA (Life
Technologies, Inc.), DNA was extracted with phenol/chloroform/isoamyl
alcohol (25:24:1) and precipitated with cold ethanol. The pellets were
washed with 70% ethanol, resuspended in loading buffer containing 85%
formamide and 1 mM NaEDTA, heated at 98 °C for
3 min, chilled on ice, and applied to an 8% polyacrylamide sequencing
gel. Autoradiographs were analyzed with a densitometer (CS-900,
Shimadzu, Kyoto, Japan). In the experiment shown in Fig. 9, DNA
was cleaved by hydroxyl radical produced by a 4-min incubation in a
mixture of 1 µM copper sulfate, 0.1 mM sodium
ascorbate, and 0.9 mM HO.
EDTA and thiourea was added to quench the reaction. In C, the dsDNA probe was incubated with H241 for 30 min, and the
complexes were then incubated further with no additions (lane
1) or with 1 mM sodium ascorbate plus 9 mM HO (lane 2); ascorbate alone (lane 3); HO alone (lane 4);
or ascorbate plus HO plus 10 µM EDTA-chelated Fe(II) (lane 5), Mg(II) (lane 6),
Zn(II) (lane 7), or Ca(II) (lane 8). Four min later,
the reaction was quenched by EDTA and thiourea. In D, the
dsDNA probe was incubated with MPC11 or 2C10 for 30 min and then for 4
min with no additions (lane 1) or with ascorbate and
HO (lanes 2 and 4) or
ascorbate plus HO plus EDTA-chelated Fe(II) (lanes 3 and 5).
) contained 10
µM Fe(II), 20 µM NaEDTA, 9 mM HO, and 1 mM sodium ascorbate (A, lanes 2 and 6; and B, lane
4). These concentrations were 0.25 (B, lane
2), 0.5 (B, lane 3), 2 (A, lane 3 and 7), and 4 (A, lanes 4 and 8) the standard. Lanes 1 and 5 in A and lane 1 in B are
uncleaved controls. Shown in C is the densitometric estimation
of the amounts of the starting DNA incubated with the hydroxyl
radical-generating mixture and H241 (
), 2C10 (), or MPC11
(
).
. The reaction time was varied
from 0 to 480 s as indicated. B, shown is the densitometric
estimation of the amounts of the starting DNA in reactions with H241
(
), 2C10 (
), and MPC11
().
100.
O in
Udenfriend's system (28) in the presence of anti-DNA
antibodies 2C10 and H241 or a control myeloma protein, MPC11. G, Maxam-Gilbert's G-reaction; G-control,
Maxam-Gilbert's G-reaction without alkaline cleavage. B,
densitometric analysis corresponds to the position of the
oligonucleotide insert described under ``Materials and
Methods.'' The ordinate represents the relative
sensitivity to the cleavage calculated as follows: relative cleavage
sensitivity = (density of cleaved ladder in the presence of
anti-DNA)/(density of cleaved ladder in the presence of MPC11). Hatched columns, H241; closed columns,
2C10.
O.
Specificity of Anti-dsDNA Antibodies
We studied
two monoclonal anti-dsDNA antibodies, 2C10 and H241, both derived from
MRL/Mp-lpr/lpr mice. In a competitive ELISA with a series of
synthetic oligonucleotides, antibody 2C10 bound to double-stranded
oligonucleotides, but did not react with single-stranded ligands in the
tested concentration range (Fig. 1A). These competitive
assays with various double-stranded polynucleotides revealed a striking
preference of 2C10 for poly(dA-dT) sequences over poly(dG-dC) or
poly(dG-dmC) sequences (Fig. 1B). In separate
experiments with other base-paired oligonucleotides, substitutions of
thymine with uracil or fluorouracil did not significantly affect the
affinity of binding to 2C10 (data not shown). Previous competitive
radioimmunoassay studies had indicated that H241 recognized a dG-dC
core in the center of a base-paired octadecanucleotide,
5`-ATATAGCGCGCGCTATAT-3`(21) .
, 5`-CGCGCATATATATATGCGCG-3` and
3`-GCGCGTATATATATACGCGC-5`; , 5`-CGCGCATAGATCTATGCGCG-3` and
3`-GCGCGTATCTAGATACGCGC-5`;
, 5`-CGCGCAGAGAGAGAGGCGCG-3`; ,
5`-CGCGCCTCTCTCTCTGCGCG-3`; , 5`-CGCGCAGAGAGAGAGGCGCG-3` and
3`-GCGCGTCTCTCTCTCCGCGC-5`. B, 2C10 was preincubated with
polynucleotides, and the mixture was added to microtiter plate wells
coated with native DNA. +, poly(dA-dT);
, poly(dG-dC);
, poly(dG-dmC). Competitive ELISA was performed as
described(21) .
P-labeled DNA almost completely at a
DNA/antibody molar ratio of 1:40 (Fig. 2A). Radioactive
oligonucleotide hardly entered the gel when the ratio was 1:80,
presumably because of the formation of larger immune complexes. H241
caused partial retardation at a ratio of 1:20 or 1:40 and complete
retardation at 1:80 (Fig. 2B). (A ratio of 1:40 with
H241 caused nearly complete retardation in some experiments (Fig. 3, lane 7).) Based on these observations, we
carried out the following experiments at a DNA/antibody ratio of 1:80
unless otherwise indicated.
Enhancement Effect of Anti-DNA Antibodies on Hydroxyl
Radical DNA Cleavage
When we began to apply chemical
footprinting assay to identification of DNA regions covered by anti-DNA
antibodies, we found, unexpectedly, a significant enhancement of DNA
cleavage by hydroxyl radical in the presence of 2C10 or H241. In these
experiments, the DNA probe in aerobic solution was preincubated with
2C10 or with the control IgG MPC11 for 30 min in PBS. Then 100
µM divalent metal ion was added, and 4 min later, the
reaction was terminated with an excess of NaEDTA and
thiourea. In the presence of antibody 2C10 and Fe(II), the DNA probe
was cleaved, while no comparable cleavage was observed with MPC11 (Fig. 4A). This cleavage resulted selectively from
hydroxyl radical production associated with oxidation of Fe(II), as
other divalent metal ions (Zn(II), Mg(II), Ca(II), or Cu(II)) did not
cleave DNA. Unlike 2C10, antibody H241 did not have a significant
effect on DNA cleavage in PBS containing 100 µM Fe(II) or
other metal ions (Fig. 4B).EDTA, HO, and
ascorbate, known as Udenfriend's system(28) , is a
convenient reagent for continuous production of hydroxyl radical. Thus,
we tested the effect of anti-DNA antibodies in the mixture of
HO and ascorbate with or without divalent metal
ions and NaEDTA (Fig. 4, C and D).
Antibody H241 markedly enhanced the cleavage of DNA by
HO and ascorbate, and this effect was not
dependent on the presence of any metal ions (Fig. 4C).
In fact, the presence of Fe(II) partially inhibited the enhancement by
H241 (Fig. 4C). In contrast, enhanced DNA cleavage by
2C10 was observed in the presence of Fe(II), ascorbate, and
HO, but not with ascorbate and
HO alone (Fig. 4D) or
ascorbate, HO, and other metals (data not
shown). The control antibody MPC11 did not enhance cleavage under any
of these conditions (Fig. 4D).Kinetics of DNA Cleavage with Anti-DNA
Antibodies
The standard reaction mixture for the following
experiments was 10 µM Fe(II), 20 µM NaEDTA, 9 mM HO, and
1 mM ascorbate. To investigate the dependence of DNA cleavage
enhancement on extent of hydroxyl radical production, the
concentrations of these four components were changed simultaneously to
give mixtures with 0.25-4.0 the standard concentrations (Fig. 5). Antibody H241 strikingly enhanced the DNA cleavage
with the standard reagent concentration, reproducibly yielding 90%
cleavage of the DNA in 4 min, as estimated from densitometric
measurement of the starting material. Antibody 2C10 also enhanced DNA
degradation, but it required higher concentrations of the reaction
mixture. At 4
the standard concentrations, most of the starting
material was degraded in 4 min even without anti-DNA antibodies.Specificity of the DNA Cleavage Enhancement
Effect
To test whether the enhanced cleavage was caused by
specific binding of the anti-DNA antibodies, self-complementary 13-mer
oligonucleotides were added to the reaction mixture to determine
whether they could inhibit the effects of 2C10 and H241. This
experiment was done at 15 °C to ensure that the complementary
oligonucleotides remained base-paired. With antibody 2C10, 0.2 and 2.0
µg of 5`-AAAATATATATTT-3` (K5) inhibited the enhancement effect of
2C10 by 49 and 83%, respectively (Fig. 7), whereas 0.2 µg of
5`-GGGGCGCGCGCCC-3` (K6) did not show significant inhibitory effect,
and 2.0 µg caused 33% inhibition. In contrast, the GC
oligonucleotide K6 was a more effective inhibitor than K5 for the
effect of H241; 0.2 and 2.0 µg of K6 caused 52 and 100% inhibition,
respectively, whereas the same concentrations of K5 caused only 6 and
32% inhibition. These results are consistent with the specificity of
2C10 and H241 described above. Thus, it is likely that the enhancement
of hydroxyl radical DNA cleavage by anti-DNA antibodies did involve the
DNA-binding sites of the two antibodies.Footprinting Protection Assay for Cro Protein
To
test whether any DNA-binding protein would enhance cleavage under the
conditions we used, we incubated the Cro repressor protein (provided by
J. Bakja, Tufts University) with a plasmid containing its recognition
sequence. The sequence was cloned into the BamHI site of
pGEM-3, the same site used for the 274-bp probe described above. The
standard hydroxyl radical-generating concentration was used. The Cro
protein protected its specific binding site from cleavage, as expected
in a footprinting assay (data not shown), consistent with previously
reported studies(30) .DNA Cleavage by Cu(I) or UV Light
To explore the
effect of other sources of hydroxyl radical on our assay system, we
tested oxidative reactions of Cu(I) and UV irradiation. For the former
reaction, Cu(II) was used in place of the very unstable Cu(I) and was
combined with HO and ascorbate. Antibody 2C10
did not enhance cleavage by this reagent; relative cleavage
sensitivities of individual sequence sites in the presence of 2C10, in
comparison with cleavage with MPC11, ranged from 0.8 and 2.2 (Fig. 9). Indeed, in the reaction with copper, the dA-dT
sequences were relatively protected by 2C10, giving a pattern opposite
to that seen in the reaction with iron. Under this experimental
condition, the uncut fraction of DNA with MPC11 was >95%. The copper
system was not tested with H241 because HO and
ascorbate alone caused marked degradation of DNA (Fig. 4C). To test the effect of UV irradiation, we
added 9 mM HO to the DNA-antibody
complex and exposed the solution to 254 nm UV light for 10 min. H241
enhanced the DNA cleavage in this reaction, but 2C10 did not (data not
shown). Thus, enhancement of DNA cleavage by 2C10 was observed only in
association with the oxidation reaction of Fe(II), whereas H241
enhanced cleavage irrespective of metal ions, and it also exhibited the
effect on UV irradiation.
)The effectiveness of this washing is consistent
with the previous demonstration of salt sensitivity of DNA binding by
antibody 2C10 (31) and other anti-DNA
autoantibodies(32) . Removal of the DNA also removes proteins
that may be associated with it in DNA-antibody complexes. O/ascorbate mixture, but not with the
copper/peroxide/ascorbate reagent or peroxide/ascorbate alone, it is
possible that antibody 2C10 selectively bound both DNA and Fe(II),
concentrating hydroxyl radical generation near the target sites. In
analogy, deliberate incorporation of a metal-binding site into an
antibody Fv domain has been proposed as a means of generating a
designed metalloenzyme(43) .O and
ascorbate without added metal ion. The presence of iron actually
reduced the antibody-mediated enhancement, whereas other ions had no
effect. Antibody H241 (and perhaps 2C10 as well) may have facilitated
cleavage by distorting the DNA structure, making target sites more
accessible. X-ray crystallographic analysis of an autoantibody to
single-stranded DNA, for example, revealed antibody stabilization of an
unpredicted conformation of oligo(dT) in the immune
complex(16) . Comparable structural data are not available for
antibodies 2C10 and H241 and their complexes with DNA. The effect is
not a property of all anti-DNA antibodies because hydroxyl radical
generation has been used effectively for protection-based footprinting
of anti-single-stranded DNA autoantibody epitopes(19) .
))))
We are grateful to Dr. Norman Krinsky for advice in
preparation of the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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